The analysis of genetically engineered variants of pp60c-src has permitted the definition of regions of the src protein (domains) that are involved in the regulations of normal c-src function and whose perturbation via mutagenesis results in the induction of the transformed phenotype. The overall aims of the experiments proposed in this application are three fold: 1) to extend our initial mutagenesis experiments and to use specific mutant forms of the c-src gene product to define the cellular proteins phosphorylated by pp60c-src, activated pp60c-src and pp60v-src; 2) to identify and characterize the cellular factors that regulate or modulate pp60c-src tyrosine protein kinase activity; and 3) to define the role of pp60c-src in the signal transduction pathway or other pathways the regulate cell growth. Using the regents generate during the first two years of the project, namely a series of transforming variants of c-src which induce various degrees of cellular transformation, we will seek to define cellular targets of the c-src protein. Three approaches will be utilized: a) cell fractionation techniques coupled with detection of tyrosine containing proteins; b) in vitro reconstitution of permeablized cells and membranes with purified proteins; c) genetic approaches that seek to identify cellular genes required for c-src function (in collaboration with M. Weber). We will continue to examine the role of Tyr 527 in the regulation of p60c-src activity and we will seek to identify cellular factors that may positively or negatively regulate pp60c-src activity, including other protein kinases. While pp60c-src has been implicated in the signal transduction pathway its position within this complicated molecular milieu remains to be defined. We propose to approach this problem by construction and utilization of a c-src replacement vector, a vector designed to suppress the resident pp60c-src and express a temperature sensitive form of the cellular src protein. Using such a vector, cell functions become dependent upon the expression f a thermolabile c-src protein whose activity can be modulated by temperature shift. Experimental manipulation of this system should provide insights as to the requirement of src function for cellular growth and mitogen/hormone responses.